To protect the host against intracellular bacterial pathogens, the innate immune system employs cell- autonomous defense pathways. Typically, these defense pathways can be divided into three steps: detection, capture and subsequent delivery of pathogens into degradative lysosomes. The first step in this cascade of events, namely the detection of intracellular bacteria, is only poorly characterized. It has been proposed that intracellular bacteria are associated with `eat-me' signals that the host cell recognizes, however, the repertoire of pathogen-associated `eat-me' signals remains largely obscure. Furthermore, many intracellular bacterial pathogens reside within pathogen-containing vacuoles (PVs), whose surrounding membranes are predominantly made out of host-derived lipids and proteins. Therefore, PVs in many ways resemble cell organelles and it is unclear how the host can recognize `organelle-like' PVs as `non-self' structures. The objective of our proposal is to define mechanisms by which the innate immune system detects PVs. Cell- autonomous immunity towards PV-resident microbes requires members of a family of Interferon (IFN)-inducible host factors known as Guanylate Binding Proteins (GBPs). It was previously shown that GBPs translocate to PVs and then solicit an array of host defense pathways towards PVs and their occupants. However, the mechanism(s) by which GBPs specifically recognize and tether to PVs are largely unknown. This proposal aims to identify and characterize PV-associated patterns that are being recognized by GBPs. One property that all bacterial PVs have in common is the presence of bacterial section systems that deliver virulence factors (`effectors') into the host cell cytosol. We hypothesize that GBPs detect the presence of bacterial secretion systems ? either directly or indirectly. Bacterial secretion systems are responsible for at least two PV- associated patterns that could be detected by GBPs: 1) the presence of conserved structural components of bacterial secretion systems exposed to the host cytosol; 2) loss of vacuolar integrity in PVs. We will test both of these models in our two Aims: in Aim1 we will probe for structural components of bacterial secretion systems that can trigger GBP targeting to PVs. We will test this hypothesis for 2 types of bacterial secretion systems: the type 4B secretion system (T4SS) and the type 3 secretion system (T3SS). Aim2 will test whether a host pathway involved in the detection of ruptured vesicles also promotes the recruitment of GBPs to PVs. Through the combined use of bacterial and mammalian genetics, cell biological and biochemical approaches, we will provide a fundamental understanding of the mechanism(s) by which GBPs sense PVs, a process required for host resistance and also linked to diseases such as sepsis.